Induction of iPS cells from Dermal Fibroblasts by High Dose HIV-Based LV Vectors in the Absence of Reprogramming Factors: Analysis of Integration Sites and Genomic Stability

Human immunodeficiency virus (HTV)-based lentivirus vectors (LV), have recently been utilised for ectopic gene transfer-mediated somatic cell reprogramming to induced pluripotent stem (iPS) cells. To date, no evidence exists suggesting multiple integration sites contribute to retroviral-mediated reprogramming. However, no association with integration site and LV-mediated reprogramming has been reported. Therefore, the biological relevance of LV integration site preferences remains unknown in iPS cell generation and remains an important safety consideration for potential clinical translational. We transduced normal human fibroblast cultures with self-inactivating (SIN) LV expressing green fluorescent protein (GFP) under control of the spleen focus forming virus promoter at an MOI of 200. 14-21d post-infection, colonies of cells with pluripotent morphology appeared in transduced cultures but not in parallel cultures lacking LV gene transfer. Cells were maintained in human embryonic stem cell (hESC) conditions. GFP +ve colony-derived cells (denoted LV-iPS cells) demonstrated ability to self-renew, expressed a mRNA and miRNA expression profile similar to pluripotent hESCs and bona fide iPS cells (but with a subset of LV-iPS-spedfic miRNAs), and formed em-bryoid bodies. Karyotyping revealed a broad range of genetic abnormalities. Comprehensive genome-wide integration site analysis revealed 23 insertion sites (preference for insertion in RefSeq genes), with altered mRNA levels observed in 60% genes located close to vector integrants selected for further analysis. We report induction of pluripotency in the complete absence of ectopic expression of iPS-reprogramming factors but with considerable transcriptional changes and insertional mutagenesis-associated detrimental changes in genomic structure and integrity induced by LV-mediated GPP gene transfer.